European Journal of Medicinal Chemistry 45 (2010) 5345e5352

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European Journal of Medicinal Chemistry

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Original article

Structureeactivity relationship studies of acridones as potential antipsoriatic agents. 2. Synthesis and antiproliferative activity of 10-substituted hydroxy-10H-acridin-9-ones against human keratinocyte growth
Aleksandar Putic, Lambert Stecher, Helge Prinz, Klaus Mller*
Institute of Pharmaceutical and Medicinal Chemistry, Westphalian Wilhelms-University, Hittorfstrae 58e62, D-48149 Mnster, Germany

a r t i c l e i n f o
Article history: Received 26 April 2010 Received in revised form 25 August 2010 Accepted 26 August 2010 Available online 20 September 2010 Keywords: Acridone derivatives Antiproliferative activity Antipsoriatic HaCaT cells Radical

a b s t r a c t
A series of 10-substituted hydroxy-10H-acridin-9-ones were synthesized and studied as potential antipsoriatic agents. The antiproliferative activity of the novel derivatives, which can be considered as azaanalogues of the antipsoriatic drug anthralin, was determined using the human keratinocyte cell line HaCaT. Structureeactivity relationships with respect to the nature of the N-substituent at the acridone scaffold were delineated. Release of lactate dehydrogenase (LDH) was used to exclude non-specic cytotoxic effects. As compared to anthralin, N-substitution of the acridone scaffold in the target compounds provided agents devoid of radical producing properties, which was documented by their ineffectiveness to interact with the free radical 2,2-diphenyl-1-picrylhydrazyl. This was in excellent agreement with the data obtained from the LDH assay in which the novel compounds did not induce membrane damage. Benzyl substitution at the 10-position yielded keratinocyte growth inhibitory activity in the low micromolar range. The most potent inhibitor of keratinocyte hyperproliferation was compound 8a having an N-methyl group and a 1,3-dihydroxy arrangement at the acridone scaffold, with an IC50 value comparable to that of anthralin. 2010 Elsevier Masson SAS. All rights reserved.

1. Introduction The acridone alkaloids constitute a small group of naturally occurring compounds produced exclusively by higher plants belonging to the order Rutales [1]. Because of their demonstrated antiproliferative activity, interest continues unabated in the synthesis of related synthetic analogues of the tetracyclic acridone alkaloid acronycine (1, Chart 1) [2e6] and 10H-acridin-9-ones possessing antitumor or other interesting biological activities [7e12]. Acridones can be considered as 10-aza-analogues of the anthrone class of topical agents against psoriasis, an inammatory and scaling skin disease, which is mainly characterized by hyperproliferation of keratinocytes [13]. Owing to the proinammatory and staining effects of antipsoriatic anthrones such as anthralin (dithranol, 1,8dihydroxy-10H-anthracen-9-one, 2), this drug is best used in hospital or day treatment centers [14], and there is need for analogues with improved therapeutic properties. Several studies have suggested that the side effects of the anthrones are associated

Abbreviations: DPPH, 2,2-diphenyl-1-picrylhydrazyl; LDH, lactate dehydrogenase; RT, room temperature; SAR, structureeactivity relationships. * Corresponding author. Tel.: 49 251 833 3324; fax: 49 251 833 2144. E-mail address: kmuller@uni-muenster.de (K. Mller). 0223-5234/$ e see front matter 2010 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.ejmech.2010.08.059

with their capability to generate oxygen radicals [15,16], and the methylene moiety at C-10 has been recognized as a key site of radical formation [17,18]. Indeed, appropriate chemical modication of the critical C-10 of the anthrone pharmacophore provided agents with diminished oxygen radical formation [19e22]. In this direction, our interest has focused on the synthesis of 10aza-analogues of anthrones in order to identify improved antipsoriatic agents that act via the keratinocyte as the primary target cell [23]. The antiproliferative activity of anthralin is intimately effected by the nature and position of the substituents at the anthrone nucleus. Both hydroxy groups peri to the keto group are required for high activity [24]. In our recent study [23] we have shown that structureeactivity relationships (SAR) for N-unsubstituted acridone derivatives did not follow those of the antipsoriatic anthrones. Surprisingly, the aza-analogue of anthralin, 1,8-dihydroxy-10H-acridin-9-one (3), was only marginally active. However, acridone 4 comprising a 1,3-dihydroxy-substitution pattern was the most active analogue, with keratinocyte growth inhibitory potency comparable to anthralin. In this communication, we expand on this previous report and discuss SAR for modication of the substituent at the acridone nitrogen atom. We have tested the novel 10-substituted acridones as inhibitors of keratinocyte hyperproliferation, which is one of the hallmarks of

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performed in the presence of sodium hydride as described by Lewis [25]. However, in both cases only the acetoacetyl-substituted acridones 11 and 12, respectively, were obtained (Scheme 4). 3. Biological assay methods In the present study, we used HaCaT keratinocytes [26] as a model for the highly proliferative epidermis in psoriasis. This rapidly dividing, non-transformed human cell line is a useful tool in identifying new topical antipsoriatic agents, and it was described as an extremely sensitive target for the antiproliferative action of the antipsoriatic drug anthralin [27]. Accordingly, we evaluated the effects of the novel acridones on the growth of cultured HaCaT cells, which was determined directly by counting the dispersed cells under a phase-contrast microscope after 48 h of treatment. Keratinocytes were also tested for their susceptibility to the action of the most potent members of this 10-substituted acridone series on plasma-membrane integrity, to conrm that inhibition of keratinocyte growth was not a result of membrane damage. This was assessed by the activity of LDH released into the culture medium [19,27]. Furthermore, free radical production by anthralin and the resulting dimerization and polymerization of the molecule is associated with the instability of the drug as well as with undesired side effects such as staining and irritation of the skin [16]. In order to compare the capability of the acridones to produce radicals by loss of a hydrogen atom with that of anthralin, we determined the extent of the N-substituted acridones to scavenge radical intermediates by measuring spectrophotometrically the decrease of DPPH concentration [28] at 520 nm. When DPPH reacts with an electron-donating test compound (i.e., a hydrogen atom donor), it is reduced to the corresponding hydrazine, whereas the primary species derived from the test compound is a free radical. 4. Results and discussion 4.1. Inhibition of keratinocyte growth In the antiproliferative study, each acridone derivative was tested for inhibitory action against HaCaT keratinocytes, as demonstrated by reduction in cell number over time as compared to control plates. The concentrations required to inhibit 50% of keratinocyte growth are shown in Table 1. From the present work, 1,3-dihydroxy-acridone 8a with an N-methyl substituent was identied as a highly potent inhibitor of keratinocyte proliferation, with an IC50 value (0.6 mM) in the

Chart 1. Structures of acronycine (1), anthralin (2), aza-analogous anthralin (3), and acridone derivative 4.

psoriasis. We also evaluated their capability to interact with a stable free radical by use of 2,2-diphenyl-1-picrylhydrazyl (DPPH). Furthermore, release of lactate dehydrogenase (LDH) was measured to exclude cytotoxic effects mediated by membrane damage.

2. Chemistry All target compounds were easily prepared from N-unsubstituted acridones. Thus, 1-hydroxyacridone (5) was N-alkylated with an appropriate alkyl halogenide and potassium carbonate according to Scheme 1. The preparation of 6a and 6d was accompanied by side products, O-alkylated 6aa and 6dd, respectively. Using benzyl bromide as an alkylating agent, also 2-benzylated 6ddd was obtained by aromatic electrophilic substitution. The structure was conrmed by NOE experiments. On irradiation at the frequency of the NCH2 protons (5.74 ppm), a positive NOE was observed for the signals at 6.96 (H-4) and 7.64 ppm (H-5), whereas on irradiation at the frequency of the ArCH2 protons (3.94 ppm), a positive NOE was observed for the signal at 7.51 (H-3) and also 14.91 ppm (OH) was detectable. In the case of 1,3-dihydroxyacridone (7), also the non-hydrogenbonded hydroxy group in 3-position was alkylated to give acridones 8aae8ee, which were then smoothly converted into the nal 1,3-dihydroxyacridones 8ae8e by ether cleavage with 48% hydrobromic acid (Scheme 2). Acridones 8aa, 8bb and 8dd were also isolated and puried. Acridones 9be9e were prepared by reaction of unsubstituted acridone (9a) with the approriate alkyl halogenides (Scheme 3). Finally, acetylation of 9a or 1-hydroxylated 5 was

Scheme 1. Reagents: (a) alkyl halogenide, K2CO3, acetone, RT or reux; (b) 48% HBr, reux.

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Scheme 4. Reagents: (a) NaH, THF, AcCl, RT, N2.

Scheme 2. Reagents: (a) alkyl halogenide, K2CO3, acetone, RT or reux; (b) 48% HBr, reux.

submicromolar range and comparable to that of standard anthralin. The naturally occurring alkaloid acronycine, which is also an N-methylated acridone and has shown activity in a large number of experimental tumor models [29], also displayed activity in the keratinocyte assay, with an IC50 value in the low micromolar range. By contrast, unsubstituted acridone (9a) was not appreciably active, whereas the corresponding N-methylated 9b displayed moderate activity. However, larger N-substituents as compared to 9b, as in 9ce9e, were detrimental for activity. In terms of SAR information, N-methylated acridone 9b without hydroxy-substituent was substantially less potent than that hydroxylated at the 1,3-positions (8a), while compound 6a with only one hydroxy-substituent at the 1-position was virtually inactive. As compared to the N-unsubstituted analogue 7, potency of 8a was dramatically improved by the 10-methyl substituent affording the most potent compound of all N-substituted acridones in this series. However, alkylation of the 3-hydroxy group (8aa, 8bb, 8dd) or only slight enlargements of the aliphatic 10-substituent as in N-ethylated 8b or in N-propylated 8c resulted in total loss of activity. In contrast to simple aliphatic substitution at the 10-position, compounds with an aliphatic chain terminated with a phenyl ring, in particular a benzyl substituent, were substantially more potent than their N-unsubstituted analogues. In the 1-hydroxy-substituted series (6ae6l), the benzylated analogues 6d and 6e as well as the methoxy-substituted analogues 6h and 6i, with an additional spacer group in the linker between the acridone scaffold and the phenyl ring, all displayed IC50 values in the low micromolar range. On the other hand, further elongation of the linker chain as in 6k and 6l with three and four spacer groups, respectively, produced inactive compounds. In the 1,3-dihydroxy series (8ae8e), N-benzylated 8d shared a comparable potency with the N-benzylated 6d and 6e. Of interest, also N-acylation of acridones 5 and 9a gave active analogues (i.e., 11 and 12), with analogue 12 being active in the low micromolar range.

There were four sets of compounds where the effect of N-methylation of the acridone scaffold could be evaluated, by comparing the corresponding NH and N-methyl derivatives. In two cases (i.e., 7, 8a and 9a, 9b), the N-methyl analogues were the more potent inhibitors of keratinocyte growth. In particular compound 8a showed a 33-fold improvement over N-unsubstituted 7. By contrast, the most potent inhibitor of our previous report, NH analogue 4, completely lost its activity upon methylation (i.e., 10). One pair of compounds (5, 6a) could not be compared, as both were inactive. 4.2. Membrane damage The release of LDH is commonly used as an indicator of plasmamembrane damage. In this assay, LDH release by the standard anthralin, which was used as a positive control, signicantly exceeded that of the vehicle control. On the other hand, the keratinocyte growth inhibitory activity of the naturally occurring acronycine and the most potent acridone derivatives was due to antiproliferative rather than non-specic cytotoxic effects, as LDH release was unchanged as compared to controls at 2-mM concentration of the test compounds (Table 1). The only exception was 6e, which caused a slight increase in LDH activity as compared to vehicle controls. This may be related to its aromatic nitro group, even though direct radical production was excluded by the DPPH assay. However, enzymatic reduction of nitro aromatic compounds gives rise to nitro radical anions that may undergo redox cycling with oxygen to generate oxygen radicals [30]. Resultant oxidative stress can produce membrane damage. 4.3. Interaction with the stable free radical DPPH Although scarcely described in the literature and only after photoirradiation and laser ash excitation of acridone, N-radical formation by hydrogen transfer to pyridine has been observed [31,32]. However, as N-substitution would prevent hydrogen transfer to a stable free radical such as DPPH, it is not surprising that with EC50 values greater than 500 mM, none of the 10-substituted acridones showed any appreciable reactivity towards DPPH. These results were in excellent agreement with our data obtained from the LDH assay in which these novel compounds were devoid of membrane-damaging properties. As a matter of fact, this is benecial as compared to anthralin, which was very effective in donating an electron to the stable free radical DPPH and, as a result, also caused desintegrity of keratinocyte membranes. The fact that the ability of the 10-substituted acridones to arrest keratinocyte growth was not mediated by non-specic redox properties or cytotoxic effects on the plasma-membrane, as observed for the antipsoriatic agent anthralin [15], opens new possibilities in the search for antipsoriatic agents.

Scheme 3. Reagents: (a) alkyl halogenide, K2CO3, acetone, reux.

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Table 1 Antiproliferative activity against HaCaT keratinocytes, lactate dehydrogenase release, and free radical interacting capability of 10-substituted 10H-acridin-9-ones.

R1
7 6

O
9 10

1 2 3 4

N R3

R2

N H

cpd 1 2 3f 4f 5f,g 6a 6aa 6b 6c 6d 6dd 6ddd 6e 6f 6g 6h 6i 6j 6k 6l 7f,h 8a 8aa 8b 8bb 8c 8d 8dd 8e 9a 9bi 9ci 9dk 9e 10 11 12
a

Substitution pattern Acronycine Anthralin 1,8-(OH)2 1,3-(OH)28eMe R1 OH, R2 H, R3 H R1 OH, R2 H, R3 Me R1 OMe, R2 H, R3 Me R1 OH, R2 H, R3 Et R1 OH, R2 H, R3 CH3(CH2)2 R1 OH, R2 H, R3 PhCH2 R1 OCH2Ph, R2 H, R3 PhCH2 1-OH-2,10-(PhCH2)2 R1 OH, R2 H, R3 4-NO2ePhCH2 R1 OH, R2 H, R3 Ph(CH2)2 R1 OH, R2 H, R3 4-OHePh(CH2)2 R1 OH, R2 H, R3 4-OMeePh(CH2)2 R1 OH, R2 H, R3 3,4-(OMe)2ePh(CH2)2 R1 OH, R2 H, R3 3,4,5-(OMe)3ePh(CH2)2 R1 OH, R2 H, R3 Ph(CH2)3 R1 OH, R2 H, R3 Ph(CH2)4 R1 OH, R2 OH, R3 H R1 OH, R2 OH, R3 Me R1 OH, R2 OMe, R3 Me R1 OH, R2 OH, R3 Et R1 OH, R2 OEt, R3 Et R1 OH, R2 OH, R3 CH3(CH2)2 R1 OH, R2 OH, R3 PhCH2 R1 OH, R2 OPhCH2, R3 PhCH2 R1 OH, R2 OH, R3 Ph(CH2)3 R1 R2 R3 H R1 R2 H, R3 Me R1 R2 H, R3 PhCH2 R1 R2 H, R3 4-OMeePhCH2 R1 R2 H, R3 3,4-(OMe)2ePhCH2 1,3-(OH)2-8,10-Me2 R1 R2 H, R3 COCH2COMe R1 OH, R2 H, R3 COCH2COMe

AAa IC50 (mM) 3.2 0.7 39.5 0.8 >50 >50 >50 >50 >50 2.5 7.5 13.0 1.8 41.5 20.1 6.3 7.0 45.5 >50 >50 19.8 0.6 >50 >50 >50 >50 3.7 >50 40.5 >50 38.9 >50 46.3 >50 >50 27.8 3.3

LDHb (mU/mL) 70.4 122.8c

e

DPPHd EC50 (mM) >500 16.7 >500 >500 >500

e e e e

71.5
e e e e e

74.9 72.1
e

>500 >500

86.7c
e e

>500 >500 >500 >500

74.6
e e e e e

e e e

74.3
e e e e

e e e e

>500 >500

76.8
e e e e e e e e e

e e e e e

>500

e e

>500 >500

74.0

>500

Antiproliferative activity against keratinocytes. IC50, concentration of test compound required for 50% inhibition of cell growth (HaCaT). Inhibition of cell growth was signicantly different with respect to that of the control, N 3, P < 0.05. b Activity of LDH (mU) release in HaCaT cells after treatment with 2 mM test compound (N 3, SD < 10%). c Values are signicantly different with respect to vehicle control (0.2% DMSO in the culture medium, 70.6 mU/mL), P < 0.05. Brij 35 (polyoxyethyleneglycol dodecyl ether)/ ultrasound was the positive control (251 mU/mL). d DPPH radical interacting capability. EC50, effective concentration of test compound (N 3, SD < 10%) required for 50% decrease in DPPH absorbance at 520 nm. NDGA (IC50 4.2 mM) and a-tocopherol (IC50 8.2 mM) were used as positive controls. e Not determined. f Ref. [23]. g Ref. [36]. h Ref. [33]. i Ref. [34]. k Ref. [8].

5. Conclusions The aim of this study was to elucidate the SAR of 10-substituted acridone analogues related to the antipsoriatic agent anthralin, to further delineate the number and position of hydroxy and other groups at the acridone scaffold that contribute to the antiproliferative activity. As already observed in our previous series of N-unsubstituted acridones [23], a 1,3-dihydroxy-substitution pattern was most benecial for potency. One member of this series, acridone 8a, with an N-methyl substituent at the 1,3-dihydroxy-substituted skeleton, was detected as a highly potent inhibitor of keratinocyte growth. With an

IC50 value of 0.6 mM, potency of the anthrone derivative anthralin was retained. As already seen with the most potent analogue of our previous series, acridone 4, which is an isomer of 8a, this N-methylated acridone was not capable of interacting with DPPH and thus producing radicals that give rise to a non-specic cytotoxic action, which would preclude a possible use in dermatology. This was in excellent agreement with the fact that in contrast to anthralin, the most potent acridones of this series did not disrupt membrane integrity. Also of interest, acylation of the acridone nitrogen atom resulted in active compounds such as 12, whose potency may further be improved with an appropriate substitution pattern. In conclusion,

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among our series of acridone analogues we were able to identify potent inhibitors of human keratinocyte growth at sufciently low concentrations to warrant investigation as antipsoriatic agents in more sophisticated systems.

6. Experimental section 6.1. Chemistry Melting points were determined with a Koer melting point apparatus and are uncorrected. 1H NMR and 13C NMR spectra were recorded with a Varian Gemini 200 (200 and 50.29 MHz, respectively) spectrometer, using tetramethylsilane as an internal standard. Fourier-transform IR spectra (KBr) were recorded on a Bio-Rad laboratories type FTS 135 spectrometer. Mass spectra were obtained in the EI mode using a MAT GCQ Finnigan instrument. Thin layer chromatography (TLC) was conducted on Merck 60 F254 precoated silica gel plates. Chromatography refers to column chromatography, which was performed on Merck silica gel (70e230 mesh). Yields have not been optimized. Elemental analyses were determined by the Microanalysis Laboratory at the University of Mnster using a Vario EL III elemental analyzer. Elemental analyses were within 0.4% of calculated values, except where stated otherwise. Acridones 6a, 6aa [25], 8a, 8aa [33], 9b, 9c [34] 9d [8], and 10 [35] were prepared according to literature methods. Acridone 9a was commercially available (SigmaeAldrich). 6.1.1. General procedure for alkylation of hydroxyacridones 6.1.1.1. 10-Ethyl-1-hydroxy-10H-acridin-9-one (6b). A mixture of 1-hydroxyacridone [36] (5, 0.20 g, 0.95 mmol), dry, nely mortared K2CO3 (0.41 g, 3 mmol) and iodoethane (3.11 g, 19.95 mmol) in dry acetone (30 mL) was stirred under reux for 5 h. Then the mixture was ltered by suction, the residue dissolved in CH2Cl2 and ltered. The combined ltrate was evaporated and the residue was puried by chromatography (SiO2; CH2Cl2/MeOH, 99/1) to provide yellow crystals; 40% yield; mp 161  C; 1H NMR (DMSO-d6) d 14.59 (s, 1H, OH), 8.34 (d, J 7.7 Hz, 1H, H-8), 7.90e7.34 (m, 4H, H-3, H-5, H-6, H-7), 7.19 (d, J 8.7 Hz, 1H, H-4), 6.64 (d, J 8.1 Hz, 1H, H-2), 4.53 (q, J 7.2 Hz, 2H, CH2CH3), 1.38 (t, J 7.1 Hz, 3H, CH2CH3); 13C NMR (DMSO-d6) d 181.3, 162.8, 142.0, 141.1, 120.0, 109.2, 136.6, 135.2, 126.0, 121.7, 115.8, 106.7, 104.4, 41.1, 34.7; FTIR 1630 cm1 (CO); MS m/z 239 (100, M). Anal. C15H13NO2 (C, H, N). 6.1.1.2. 1-Hydroxy-10-propyl-10H-acridin-9-one (6c). The title compound was obtained from 5 and 1-iodopropane using the same procedure as for 6b, but the mixture was reuxed for 24 h. Purication by chromatography (SiO2; CH2Cl2) gave golden yellow crystals; 63% yield; mp 92  C; 1H NMR (DMSO-d6) d 14.59 (s, 1H), 8.32 (d, J 7.6 Hz, 1H), 7.87e7.32 (m, 4H), 7.15 (d, J 8.9 Hz, 1H), 6.62 (d, J 8.0 Hz, 1H), 4.37 (t, J 8.1 Hz, 2H), 1.83e1.72 (m, 2H), 1.05 (t, J 7.3 Hz, 3H); FTIR 1634 cm1 (CO); MS m/z 253 (100, M). Anal. C16H15NO2 (C, H, N). 6.1.1.3. 10-Benzyl-1-hydroxy-10H-acridin-9-one (6d). The title compound was obtained from 5 and benzyl bromide using the same procedure as for 6b, but the mixture was reuxed for 24 h. Purication by chromatography (SiO2; CH2Cl2) gave gold-colored crystals; 77% yield; mp 176  C; 1H NMR (DMSO-d6) d 14.50 (s, 1H, OH), 8.30 (d, J 7.3 Hz, 1H, H-8), 7.89e7.12 (m, 9H, Ar), 6.95 (d, J 8.8 Hz, 1H, H-4), 6.65 (d, J 8.0 Hz, 1H, H-2), 5.79 (s, 2H, CH2); 13 C NMR (DMSO-d6) d 181.5, 162.7, 142.9, 142.0, 136.5, 135.8, 135.1, 128.7 (2C), 127.2, 125.8, 125.7 (2C), 122.0, 120.1, 116.1, 109.1, 107.1, 104.9, 49.6; FTIR 1630 cm1 (CO); MS m/z 301 (70, M), 91 (100). Anal. C20H15NO2 (C, H, N).

6.1.1.4. 10-Benzyl-1-benzyloxy-10H-acridin-9-one (6dd). The title compound was obtained as a side product of 6d. Purication by chromatography (SiO2; CH2Cl2) gave gold-colored crystals; 5% yield; mp 179  C; 1H NMR (DMSO-d6) d 8.32 (d, J 6.7 Hz, 1H, H-8), 7.73e7.13 (m, 14H, Ar), 7.05 (d, J 8.7 Hz, 1H, H-4), 6.91 (d, J 8.1 Hz, 1H, H-2), 5.68 (s, 2H, OCH2), 5.26 (s, 2H, NCH2); FTIR 1632 cm1 (CO); MS m/z 391 (100, M). Anal. C27H21NO2 (C, H, N). 6.1.1.5. 2,10-Dibenzyl-1-hydroxy-10H-acridin-9-one (6ddd). The title compound was obtained as a side product of 6d, when the mixture was reuxed for 50 h. Purication by chromatography (SiO2; CH2Cl2) gave yellow crystals; 13% yield; mp 191  C; 1H NMR (DMSO-d6) d 14.91 (s, 1H, OH), 8.39 (dd, J 8.1, 1.7 Hz, 1H, H-8), 7.83e7.10 (m, 14H, Ar), 6.96 (d, J 8.9 Hz, 1H, H-4), 5.74 (s, 2H, NCH2Ar), 3.94 (s, 2H, ArCH2Ar); FTIR 1622 cm1 (CO); MS m/z 391 (100, M). Anal. C27H21NO2 (C, H, N). 6.1.1.6. 1-Hydroxy-10-(4-nitrobenzyl)-10H-acridin-9-one (6e). The title compound was obtained from 5 and 4-nitrobenzyl bromide using the same procedure as for 6b, but the mixture was reuxed for 9 h. Purication by chromatography (SiO2; CH2Cl2) gave yellow crystals; 46% yield; mp 282  C; 1H NMR (DMSO-d6) d 14.59 (s, 1H, OH), 8.40 (dd, J 1.4, 8.1 Hz, 1H, H-8), 8.18 (d, J 8.5 Hz, 2H, H-30 , H50 ), 7.82 (t, J 7.3 Hz, 1H, H-6), 7.66e7.39 (m, 3H, H-3, H-5, H-7), 7.41 (d, J 8.5 Hz, 2H, H-20 , H-60 ), 6.96 (d, J 8.7 Hz, 1H, H-4), 6.68 (d, J 8.0 Hz, 1H, H-2), 5.94 (s, 2H, CH2); FTIR 1638 cm1 (CO); MS m/z 346 (100, M). Anal. C20H14N2O4 (C, H, N). 6.1.1.7. 1-Hydroxy-10-(2-phenylethyl)-10H-acridin-9-one (6f). The title compound was obtained from 5 and 2-bromoethylbenzene using the same procedure as for 6b, but the mixture was reuxed for 24 h. Purication by chromatography (SiO2; CH2Cl2/MeOH, 98/ 2) gave yellow crystals; 12% yield; mp 162  C; 1H NMR (DMSO-d6) d 14.59 (s, 1H, OH), 8.36 (d, J 7.9 Hz, 1H, H-8), 7.89e7.20 (m, 10H, Ar), 6.66 (d, J 7.8 Hz, 1H, H-2), 4.66 (t, J 8.0 Hz, 2H, NCH2CH2), 3.10 (t, J 8.1 Hz, 2H, NCH2CH2); FTIR 1630 cm1 (CO); MS m/z 315 (91, M), 224 (100). Anal. C21H17NO2 (C, H, N). 6.1.1.8. 1-Hydroxy-10-[2-(4-hydroxyphenyl)ethyl]-10H-acridin-9one (6g). The title compound was obtained from 6h using the same procedure as for 8b. Purication by chromatography (SiO2; CH2Cl2) gave yellow crystals; 43% yield; mp 230  C; 1H NMR (DMSO-d6) d 14.59 (s, 1H), 9.28 (s, br, 1H), 8.35 (d, J 7.7 Hz, 1H), 7.89e6.64 (m, 10H), 4.58 (t, J 8.0 Hz, 2H), 2.98 (t, J 8.0 Hz, 2H); FTIR 3329, 1637 cm1 (CO); MS m/z 331 (48, M), 212 (100). Anal. C21H17NO3 (C, H, N). 6.1.1.9. 1-Hydroxy-10-[2-(4-methoxyphenyl)ethyl]-10H-acridin-9one (6h). The title compound was obtained from 5 and 1-(2-bromoethyl)-4-methoxybenzene [37] using the same procedure as for 6b, but the mixture was reuxed for 24 h. Purication by chromatography (SiO2; twice: hexane, then CH2Cl2) gave yellow crystals; 24% yield; mp 126  C; 1H NMR (DMSO-d6) d 14.59 (s, 1H), 8.35 (d, J 7.8 Hz, 1H), 7.88 (d, J 3.1 Hz, 1H), 7.71 (t, J 8.3 Hz, 1H), 7.39e7.33 (m, 2H), 7.35 (d, J 8.6 Hz, 2H), 7.21 (d, J 8.5 Hz, 1H), 6.88 (d, J 8.4 Hz, 2H), 6.66 (d, J 8.0 Hz, 1H), 4.60 (t, J 8.1 Hz, 2H), 3.72 (s, 3H), 3.03 (t, J 7.9 Hz, 2H); 13C NMR (DMSO-d6) d 181.3, 162.8, 158.1, 142.1, 141.2, 136.6, 135.2, 130.0 (2C), 129.5, 125.9, 121.8, 120.0, 115.9, 113.9 (2C), 109.0, 106.9, 104.6, 55.0, 47.4, 31.6; FTIR 1633 cm1 (CO); MS m/z 345 (20, M), 134 (100). Anal. C22H19NO3 (C, H, N). 6.1.1.10. 1-Hydroxy-10-[2-(3,4-dimethoxyphenyl)ethyl]-10H-acridin9-one (6i). The title compound was obtained from 5 and 1-(2-bromoethyl)-3,4-dimethoxybenzene [38] using the same procedure as

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for 6b, but the mixture was reuxed for 24 h. Purication by chromatography (SiO2; twice: CH2Cl2, then CH2Cl2/MeOH, 98/2) gave yellow-orange crystals; 28% yield; mp 125  C; 1H NMR (DMSO-d6) d 14.59 (s, 1H), 8.35 (d, J 7.7 Hz, 1H), 7.89e6.64 (m, 9H), 4.64 (t, J 7.8 Hz, 2H), 3.74 (s, 3H), 3.70 (s, 3H), 3.02 (t, J 7.9 Hz, 2H); FTIR 1635 cm1 (CO); MS m/z 375 (17, M), 164 (100). Anal. C23H21NO4 (C, H, N). 6.1.1.11. 1-Hydroxy-10-[2-(3,4,5-trimethoxyphenyl)ethyl]-10H-acridin-9-one (6j). The title compound was obtained from 5 and 1-(2bromoethyl)-3,4,5-trimethoxybenzene [38] using the same procedure as for 6b, but the mixture was reuxed for 24 h. Purication by chromatography (SiO2; twice: CH2Cl2, then CH2Cl2/MeOH, 98/2) gave yellow-orange crystals; 5% yield; mp 156  C; 1H NMR (DMSOd6) d 14.60 (s, 1H), 8.35 (d, J 7.9 Hz, 1H), 7.88e6.61 (m, 8H), 4.71 (t, J 7.5 Hz, 2H), 3.72 (s, 6H), 3.58 (s, 3H), 3.02 (t, J 7.6 Hz, 2H); FTIR 1630 cm1 (CO); MS m/z 405 (19, M), 194 (100). Anal. C24H23NO5 (C, H, N). 6.1.1.12. 1-Hydroxy-10-(3-phenylpropyl)-10H-acridin-9-one (6k). The title compound was obtained from 5 and 3-bromopropylbenzene using the same procedure as for 6b, but the mixture was reuxed for 24 h. Purication by chromatography (SiO2; CH2Cl2) gave yellow crystals; 55% yield; mp 120  C; 1H NMR (DMSO-d6) d 14.56 (s, 1H, OH), 8.31 (dd, J 1.7, 8.1 Hz, 1H, H-8), 7.82e7.20 (m, 9H, Ar), 6.99 (d, J 8.6 Hz, 1H, H-4), 6.60 (d, J 7.9 Hz, 1H, H-2), 4.39 (t, J 8.0 Hz, 2H, NCH2CH2CH2), 2.84 (t, J 7.7 Hz, 2H, NCH2CH2CH2), 2.08e2.01 (m, 2H, NCH2CH2CH2); FTIR 1620 cm1 (CO); MS m/z 329 (69, M), 224 (100). Anal. C22H19NO2 (C, H, N). 6.1.1.13. 1-Hydroxy-10-(4-phenylbutyl)-10H-acridin-9-one (6l). The title compound was obtained from 5 and 4-bromobutylbenzene [39] using the same procedure as for 6b, but the mixture was reuxed for 24 h. Purication by chromatography (SiO2; hexane/ CH2Cl2, 85/15) gave yellow crystals; 90% yield; mp 108  C; 1H NMR (DMSO-d6) d 14.58 (s, 1H), 8.31 (dd, J 1.5, 8.0 Hz, 1H), 7.83e7.15 (m, 9H), 7.07 (d, J 8.7 Hz, 1H), 6.61 (d, J 7.9 Hz, 1H), 4.42 (t, J 8.3 Hz, 2H), 2.66 (t, J 6.8 Hz, 2H), 1.80e1.76 (m, 4H); FTIR 1628 cm1 (CO); MS m/z 343 (100, M). Anal. C23H21NO2 (C, H, N). 6.1.1.14. 3-Ethoxy-10-ethyl-1-hydroxy-10H-acridin-9-one (8bb). The title compound was obtained from 1,3-dihydroxyacridone (7) [33] and iodoethane using the same procedure as for 6b, but the mixture was reuxed for 24 h. Purication by chromatography (SiO2; CH2Cl2/MeOH, 98/2) gave yellow crystals; 68% yield; mp 170e172  C; 1H NMR (DMSO-d6) d 14.92 (s, 1H, OH), 8.26 (d, J 8.2 Hz, 1H, H-8), 7.81e7.78 (m, 2H, H-5, H-6), 7.31 (dd, J 8.0, 5.8, 2.1 Hz, 1H, H-7), 6.47 (d, J 2.1 Hz, 1H, H-4), 6.22 (d, J 1.9 Hz, 1H, H-2), 4.42 (q, J 7.0 Hz, 2H, OCH2CH3), 4.15 (q, J 6.9 Hz, 2H, NCH2CH3), 1.39e1.30 (m, 6H, OCH2CH3, NCH2CH3); FTIR 1639 cm1 (CO); MS m/z 283 (100, M). Anal. C17H17NO3 (C, H, N). 6.1.1.15. 10-Benzyl-3-benzyloxy-1-hydroxy-10H-acridin-9-one (8dd). The title compound was obtained from 7 and benzyl bromide using the same procedure as for 6b, but the mixture was stirred at RT for 24 h. Purication by chromatography (SiO2; hexane/CH2Cl2, 1/1) and recrystallization from CH2Cl2/MeOH (1/1) gave pale-yellow crystals; 20% yield; mp 224e225  C; 1H NMR (DMSO-d6) d 14.66 (s, 1H), 8.17 (d, J 8.1 Hz, 1H), 7.86e7.10 (m, 14H), 6.48 (s, 1H), 5.21 (s, 2H), 4.30 (s, 2H); FTIR 3345, 1635 cm1 (CO); MS m/z 407 (75, M), 316 (100). Anal. C27H21NO3 (C, H, N). 6.1.1.16. 10-(3,4-Dimethoxybenzyl)-10H-acridin-9-one (9e). The title compound was obtained from acridone (9a) and 3,4-dimethoxybenzyl

bromide [40] using the same procedure as for 6b, but the mixture was reuxed for 24 h. Purication by chromatography (SiO2; CH2Cl2) gave orange crystals; 20% yield; mp 215e216  C; 1H NMR (CDCl3) d 8.60 (dd, J 8.0 Hz, J 1.8 Hz, 2H, H-1, H-8), 7.76e7.54 (m, 2H, H-3, H-6), 7.46e7.16 (m, 4H, H-2, H-4, H-5, H-7), 7.02e6.74 (m, 2H, H-20 , H-50 ), 6.46e6.28 (m, 1H, H-60 ), 5.63 (s, 2H, NCH2), 4.07 (s, 3H, OCH3), 3.94 (s, 3H, OCH3); FTIR 3345, 1634 cm1 (CO); MS m/z 345 (38, M), 151 (100). Anal. C22H19NO3 (C, H, N). 6.1.2. General procedure for the cleavage of ethers 6.1.2.1. 10-Ethyl-1,3-dihydroxy-10H-acridin-9-one (8b). A solution of 8bb (0.62 g, 2.20 mmol) in 48% HBr (30 mL) was reuxed for 3 h. Then the solution was allowed to cool to RT. The precipitating crystals were ltered by suction, washed with water (2 100 mL), dissolved in CH2Cl2 (100 mL), and the aqueous phase was extracted with CH2Cl2 (2 100 mL). The combined organic phase was dried over Na2SO4, evaporated, and the residue puried by chromatography (SiO2; CH2Cl2/MeOH, 98/2) to provide yellow-green crystals; 94% yield; mp 293  C; 1H NMR (DMSO-d6) d 14.90 (s, 1H, 1-OH), 10.65 (s, 1H, 3-OH), 8.28 (d, J 7.4 Hz, 1H, H-8), 7.85e7.19 (m, 3H, H-5, H-6, H-7), 6.43 (d, J 1.7 Hz, 1H, H-4), 6.11 (d, J 1.7 Hz, 1H, H-2), 4.37 (q, J 6.9 Hz, 2H, CH2CH3), 1.36 (t, J 7.0 Hz, 3H, CH2CH3); 13C NMR (DMSO-d6) d 179.9, 165.0, 164.9, 143.6, 140.9, 120.0, 103.7, 134.4, 125.8, 121.3, 115.3, 95.9, 90.8, 41.0, 11.9; FTIR 1655 cm1 (CO); MS m/z 255 (100, M). Anal. C15H13NO3 (C, H, N). 6.1.2.2. 1,3-Dihydroxy-10-propyl-10H-acridin-9-one (8c). The title compound was obtained from 7 [33] and 1-iodopropane using the same procedure as for 6b, but the mixture was reuxed for 24 h. The crude product 8cc was then processed as described for 8b. Purication by chromatography (SiO2; CH2Cl2/MeOH, 98/2) gave yellow crystals; 29% yield; mp 213  C; 1H NMR (DMSO-d6) d 14.89 (s, 1H), 10.65 (s, 1H), 8.25 (d, J 8.2 Hz, 1H), 7.83e7.71 (m, 2H), 7.30 (t, J 7.0 Hz, 1H), 6.38 (d, J 1.7 Hz, 1H), 6.09 (d, J 1.6 Hz, 1H), 4.20 (t, J 7.8 Hz, 2H), 1.81e1.69 (m, 2H), 1.05 (t, J 7.3 Hz, 3H); FTIR 1639 cm1 (CO); MS m/z 269 (82, M), 227 (100). Anal. C16H15NO3 (C, H, N). 6.1.2.3. 10-Benzyl-1,3-dihydroxy-10H-acridin-9-one (8d). The title compound was obtained from 8dd as described for 8b, but the mixture was reuxed for 9 h. Purication by chromatography (SiO2; CH2Cl2) gave yellow crystals; 13% yield; mp 233  C; 1H NMR (DMSO-d6) d 14.50 (s, 1H), 10.69 (s, br, 1H), 8.14 (dd, J 8.1, 1.2 Hz, 1H), 7.82e7.07 (m, 9H), 6.19 (s, 1H), 4.21 (s, 2H); FTIR 3402, 1650 cm1 (CO); MS m/z 317 (100, M). Anal. C20H15NO2 (C, H, N). 6.1.2.4. 1,3-Dihydroxy-10-(3-phenylpropyl)-10H-acridin-9-one (8e). The title compound was obtained from 7 [33] and 3-bromopropylbenzene using the same procedure as for 6b, but the mixture was reuxed for 48 h. The crude product 8ee was then processed as described for 8b. Purication by chromatography (SiO2; CH2Cl2/ MeOH, 99/1) gave yellow crystals; 22% yield; mp 204  C; 1H NMR (DMSO-d6) d 14.89 (s, 1H), 10.69 (s, 1H), 8.26 (dd, J 8.0, 1.3 Hz, 1H), 7.75 (t, J 7.2 Hz, 1H), 7.60 (d, J 8.7 Hz, 1H), 7.34e7.17 (m, 6H), 6.36 (d, J 1.3 Hz,1H), 6.10 (d, J 1.4 Hz,1H), 4.27 (t, J 7.2 Hz, 2H), 2.84 (t, J 7.6 Hz, 2H), 2.07e2.03 (m, 2H); 13C NMR (DMSO-d6) d 179.3, 165.0 (2C), 143.9, 141.1, 141.0, 134.4, 128.4 (2C), 128.2 (2C), 126.0, 125.7, 121.4, 119.9, 115.2, 103.7, 95.9, 90.6, 45.6, 31.9, 27.9; FTIR 1638 cm1 (CO); MS m/z 345 (99, M), 241 (100). Anal. C22H19NO3 (C, H, N). 6.1.3. 1-(9-Oxo-9H-acridin-10-yl)butane-1,3-dione (11) To a solution of acridone (9a, 1.0 g, 5.1 mmol) in dimethylformamide (70 mL) was added NaH (60%, 0.19 g) under N2. The mixture was stirred at RT for 1 h. Then acetyl chloride (2 mL, 28.0 mmol) was added. Three further successive additions of NaH

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followed by acetyl chloride were made over 48 h. The mixture was poured into ice-water (120 mL) and the residue ltered by suction. Purication by chromatography (SiO2; CH2Cl2) gave pale-yellow crystals; 26% yield; mp 121  C; 1H NMR (DMSO-d6) d 8.34e7.36 (m, 8H), 5.84 (d, J 2.6 Hz, 2H), 2.15 (s, 3H); FTIR 1764 (NeCO), 1608 cm1 (CO); MS m/z 279 (55, M), 195 (100). Anal. C17H13NO3 (C, H, N). 6.1.4. 1-(1-Hydroxy-9-oxo-9H-acridin-10-yl)butane-1,3-dione (12) The title compound was obtained from 5 [36] (0.5 g, 2.4 mmol) in THF (40 mL) as described for 11. The reaction mixture was treated with water (80 mL) and extracted with CH2Cl2 (3 30 mL). The combined organic phase was dried over Na2SO4, evaporated, and the resulting red-brown oil was puried by chromatography (SiO2; CH2Cl2/MeOH, 99/1) to afford yellow crystals; 12% yield; mp 149  C; 1 H NMR (CD3OD) d 13.98 (s, 1H, OH), 8.34e6.51 (m, 7H, Ar), 5.82 (d, J 2.6 Hz, 2H, CH2), 2.16 (s, 3H, CH3); FTIR 1774 (NeCO), 1637 cm1 (CO); MS m/z 295 (49, M), 211 (100). Anal. C17H13NO4 (C, H, N). 6.2. Biological assay methods 6.2.1. Keratinocyte culture and determination of cell growth HaCaT cells [26] were cultivated and the cell proliferation assay was performed as described [41]. After 48 h of incubation, cell growth was determined by enumerating the dispersed cells by phase-contrast microscopy. Inhibition was calculated by the comparison of the mean values of the test compound (N 3) with the control (N 6e8) activity: (1 - test compound/control) 100. Inhibition was statistically signicant compared to that of the control (Students t-test; P < 0.05). IC50 values were obtained by nonlinear regression. 6.2.2. Lactate dehydrogenase release The assay was performed as described [19,27]. HaCaT cells were incubated with the test compounds (2 mM) for 4 h at 37  C. Extracellular LDH activity was measured using the UV method with pyruvate and NADH and is expressed in mU/ml. Appropriate controls with the vehicle were performed (P < 0.05; N 3, SD < 10%). Brij 35 (polyoxyethyleneglycol dodecyl ether)/ultrasound was the positive control. 6.2.3. Determination of DPPH radical interacting capability The assay was performed as described [23]. The capability to interact with DPPH radical (%) was calculated by the comparison of the mean values of the test compound (N 3) with those of the control (N 8): (1absorbance of test compound/absorbance of control) 100. Results were expressed as EC50, the effective concentration of the test compound required for 50% decrease in DPPH absorbance at 520 nm. Values were obtained by nonlinear regression. Appendix. Supplementary data Supplementary data associated with this article can be found in the online version, at doi:10.1016/j.ejmech.2010.08.059. References
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